Proximity-dependent proteomics of the Chlamydia trachomatis inclusion membrane reveals functional interactions with endoplasmic reticulum exit sites
Fig 1
In situ proteomic labeling of the C. trachomatis inclusion membrane.
(A) Plasmid used to transform C. trachomatis L2 and localize APEX2 to the inclusion membrane. IncB-APEX2 fusion expression was under the control of a tetracycline inducible promoter. Flag epitope tag was added between IncA and APEX2. (B) Schematic for APEX2 localization and biotinylation reaction. Cells were infected with C. trachomatis expressing IncB-APEX2 for 8, 16, or 24 hours, expression was induced by anhydrotetracycline, and incubation with biotin-phenol and hydrogen peroxide for 1-minute catalyzed biotinylation of proteins within 20 nm of APEX2. Biotinylated proteins were enriched using streptavidin coated agarose resin and relative abundance estimated using mass spectrometry-based proteomics. (C) Western blot analysis of cells infected for 16 hours with Chlamydia expressing IncB-APEX2 (first four columns) or untagged APEX2 (last column). Blots were probed with streptavidin-HRP to detect biotinylated proteins. Anti-beta-actin and anti MOMP antibodies were used as human and Chlamydia loading controls, respectively. BP, biotin-phenol; ATc, anhydrotetracycline. (D) Immunofluorescence microscopy of cells infected with C. trachomatis IncB-APEX2 and after 1 min inclusion membrane protein labeling at 8, 16, and 24 hpi. Representative images are shown. Biotin labeled proteins were identified by streptavidin-Alexa 488, Chlamydia were labeled with an anti-MOMP antibody, DNA labeled with DAPI. Single channel images are displayed in inverted grayscale. Merged panels display all three-color channels. Scale bars = 16 μm.